Iran J Reprod Med Vol. 13. No. 8. pp: 489-494, August 2015 Original article

The adverse effects of the methoxsalen and ultraviolent A radiation on spermatogenesis in mice

Mona Farhadi1 Ph.D., Homa Mohseni Kouchesfahani3 Ph.D., Abass Shockravi2 Ph.D., MosaeebForoozanfar3 Ph.D., Kazem Parivar3 Ph.D.

1. Department of Mcrobiology, Abstract Karaj Branch, Islamic Azad University, Karaj, Iran. Background: Different investigation showed that 5-methoxypsoralen and 8- 2. Faculty of Chemistry, Kharazmi methoxypsoralen reduce birth rates in the rats. University, Tehran, Iran. Objective: In this study we worked out the effect of methoxsalen together with 3. Department of Biology, ultraviolent A (UVA) radiation on mature Balb/C mice spermatogenesis. Kharazmi University, Tehran, Iran. Materials and Methods: The LD50 standard was determined 160 mg/kg and the UVA dose which causes erythema was calculated 0.046 J/cm2. A sub-lethal dose of 80 mg/kg of methoxsalen solution was injected intrapritoneally to mature mice and after one hour they were exposed to UVA radiation for 20 minutes. Experiments

applied included methoxsalen alone, methoxsalen with UVA, UVA alone, sham group (a group received Tween 80), and control group (N=6). In all experimental groups except UVA alone group, injections were carried out, during two consecutive weeks. Serial cross sections (5 µm thickness) were prepared for morphological and

histological studies. Tunica albuginea diameter, and number of type A and type B spermatogonia and histological investigation of the testes were measured. Results: Microscopical and statistical analyses showed significant anomalies among the experimental groups compared to control and sham group. These anomalies

included decrease the body weight; increase the relative testis weight; and decrease the number of spermapogonia (type A and B), primary spermatocytes, spermatids and sperms in experimental groups I and II compared to control group. Our results showed the number of spermatozoa in experimental group I was 22.6±2.12, in

Corresponding Author: experimental group II was 33.6±2.05 and in control group was 44.3±2.77 (p<0.05). Mona Farhadi, Department of Moreover in some experimental groups (I and II) shrinkage of seminiferous tubules Mcrobiology, Karaj Branch, and release of primary spermatocyte and spermatids were observed to the lumen of Islamic Azad University, Karaj, them. Iran. P.O.Box: 31485-313 Email: [email protected] Conclusion: It is concluded from the results of this work that treatment with Tel: (+98) 9122467389 methoxsalen with UVA can damage and disorganize seminiferous tubules and decrease spermatogenic cells.

Received: 24 November 2013 Revised: 16 February 2015 Key words: Methoxsalen, UVA, Abnormality, Spermatogenesis. Accepted: 11 May 2015

Introduction known to be toxic to a wide range of organisms (7). In the last three decades soralens are a class of three rings, researchers were able to show that psoralens heterocyclic and planar compounds inhibit epidermal cell proliferation after UV P that occur naturally as secondary light irradiation (6). metabolites in various plant species (1, 2, 3, It has been shown that methoxsalene has 4) Methoxsalen (8-methoxypsoralen) together toxic effects on reproductive and with ultraviolet A (UVA) long-wave therapy developmental processes (8). Studies by (PUVA) is known to be a human carcinogen Diawara et al. (1997) showed that, oral based on sufficient evidence of administration of methoxsalen to male and carcinogenicity from studies in humans. In female rats reduces birth rate significantly. photo chemotherapy methoxsalen and UVA Also, 200 to 400 mg/Kg dosage of (320-400 nm) are used for treatment of some methoxsalen could reduce animal weight and epidermal proliferative disorders such as could change the histology of the liver, testes psoriasis and vitiligo (5, 6). Psoralens are and adrenal (9). In 2013 it was shown that Farhadi et al methoxsalen decreased mean level of methoxsalen with UVA and PUVA, while estrogen and progesterone significantly and it experimental group III were under UVA only. significantly reduced the number and diameter Injections were applied for two consecutive of corpus luteum and number of growing weeks. The animals of experimental groups (I, follicles (10). Ruta graveolens (RG) is one of II, III) were sacrificed 48 hours after the last the species of herbal medicinal families that injection, and their testes were removed and use for male contraception (11, 12). Long term fixed in Bouins’s fixative and embedding with administration of RG can decrease the weight paraffin. Then serial cross sections (5 µm of genital organs, sperm motility and sexual thickness) were prepared and slides were behavior in rat (13). RG plus 8-methoxy stained with H & E solutions for morphological psoralen (sodab) caused weight gain in and histological studies (15). The following observations were recorded: initial and final testicle and epydidim (14). These findings male body weights, testes weights, Tunica showed that psoralen could affect the male albuginea diameter, and number of and female reproductive systems. Despite of spermatogonia and histological investigation the long term application of furocoumarines of the testes. Morphometric measurements (methoxsalen, xanthotoxin, bergapten, were applied by light microscopy. Tubule isopimpinellin) in traditional medicine, the diameters of 100 nearly round seminiferous teratogenicity of methoxsalen alone or tubules were measured for each testis using together with UV radiation is not clear yet. an ocular micrometer at 250X. Diameter This study was applied to investigate the averaged for each rat for statistical analysis probable teratogenicity of methoxsalen on (16). All animal-related protocols were unmated male mature Balb/C mice. approved by Ethical Committee at Kharazmi University, Tehran, Iran. Materials and methods Statistical analysis In an experimental study in Kharazmi In all experiments, data were analyzed using University we used Methoxsalen with formula SPSS (Statistical Package for the Social of 9-methoxy-7H-furo [3, 2-g] [1]-benzopyran- Sciences, SPSS Inc., Chicago, IL, USA) 7-one that was purchased from Sigma Aldrich software, and one way ANOVA statistical (Germany) and Tween 80 was used as method. For each variable, means were solvent. The UVA source consisted of a calculated at the 5% level using LSD test. L40/79k (Osram) lamp emitting 7.632×10 (w/cm) at 50 cm distance. The UVA dose Results which causes erythema is 0.046 J/cm2. Male Balb/C mice were obtain from Pasture The results showed that body weight Institute (Experimental Animal Keeping reduced significantly in all experimental Center, Tehran, Iran) and were grown in the groups compared to the control group. animal room at temperature of 22-23°C, 50- Relative testes weight and the thickness of 55% humidity and lighting cycle of 12-h tunica albuginea, was significantly increased light/12-h dark. The animals were randomly (Figure 1) in experimental groups I and II, divided in three experimental, sham and while the testis volume and its length and control groups which all were unmated mature diameter did not show significant changes (Table I). The number of type A and B Balb/C mice (65 days old), 6 in each group. spermatogonia, primary spermatocytes, The LD50 standard was determined 160 spermatids and sperms were significantly mg/kg body weight and the UVA dose which reduced in all experimental groups, compared causes erythema was applied (0.046 g/cm). A to control group (Table II). In addition, PUVA sub-lethal dose of 80 mg/kg methoxsalen experience (experimental group I) and solution was injected intrapritoneally to mature methoxsalen alone expeience (experimental mice and after one hour the mice were group II) showed shrinkage of seminiferous exposed to UVA radiation for 20 minutes. tubules, release of primary spermatocytes and Experimental group I received methoxsalen sperms into the lumen, and disorganized only; experimental group II received seminiferous tubules (Figure 2).

490 Iranian Journal of Reproductive Medicine Vol. 13. No. 8. pp: 489-494, August 2015 Effect of methoxsalen on spermatogenesis

Table I. Results of the effects of methoxsalen on the testes (mean±SE)

Groups Body weight * Relative testis weight** Thickness of Tunica albuginea µm Controls 2.35±0.57 0.774±0.03 13.7±0.63

Sham 2.17±0.71 0.779±0.03 14.74±0.66

Methoxalen (experimental group I) -12.51±0.71 0.885±0.03 21.5±0.82

Methoxalen with UVA (experimental group II) -11.9±1.18 0.883±0.03 31.62±0.84

UVA (experimental group III) -1.28±0.57 0.771±0.02 19.7±0.77

* Body weight gain= (w2-w1)/w1×100: w1=animal weight before experiment, w2=animal weight after experiment and SE=standard error ** Relative testis weight =(testis weight/body weight)×100 b: Statistical difference to control group (p<0.05) UVA: Ultraviolent A

Table II. Results of the effects of methoxsalen on the number of spermatogenic cells (mean±SE)

No. of spermatogonia No. of spermatogonia No. of primary No. of No. of Groups Type A Type B spermatocyte spermatid spermatozoid Controls 2.4±0.12 13.52±0.67 18.2±0.82 66.5±2.81 44.3±2.77

Sham 2.2±0.11 13.22±0.65 17.4±0.79 64.9±2.63 44.6±2.76

Methoxalen (experimental group I) 1.44±0.13 9.6±0.715 15.14±0.73 39.9±2.67 22.6±2.12

Methoxalen with UVA(experimental group II) 1.28±0.11 7.64±0.497 12.04±0.71 46.8±2.84 33.6±2.05

UVA (experimental group III) 1.52±0.12 10.96±0.67 16.7±0.71 49.1±1.54 38.5±2.56

b: Statistical difference to control group (p<0.05). UVA: Ultraviolent A

Figure 1. Photomicrograph of tunica albuginea of testis. Control (left), experimental: methoxsalen with UVA (right). The increase of tunica albuginea diameter (2500X)

Figure 2. Photomicrograph of seminiferous tubules of testis. Control (left) experimental: methoxsalen with UVA (right). The shrank seminiferous tubule with empty lumen of sperm (1000X)

Iranian Journal of Reproductive Medicine Vol. 13. No. 8. pp: 489-494, August 2015 491 Farhadi et al

Discussion as a results of anti-apoptotic and tumor induction effects of methoxsalen. Whereas, 8-methoxypesoralen (methoxsalen) with this increase in experimental group III UVA (PUVA) has recovery effect on several compared to control group was not significant; autoimmune diseases including psoriatic one reason for this result could be because of arthritis, skin disorders and also reproductive accumulative side effects of UVA together system (8, 16). Despite the treatment effects with methoxsalen that needs further of PUVA, it has photo toxicity, mutagenic and investigation. carcinogenic effects on various physiological Methoxsalen induces liver enzyme mRNA, pathways and cell components (15, 17, 18). cytochrome P4501A1 and UDP- Different studies indicated, various byproducts glucuronosyltransferase1A6, suggesting of methoxsalen could have toxicity effects on enhancement of metabolism of estrogen. cell membrane or nucleic acids (19). Our Thus, methoxsalen treatment may explain the findings showed, methoxsalen can affect male reproductive toxicity including reduction of reproductive organs; toxicity of methoxsalen ovarian follicular function and ovulation, effect lead to decrease in the number of on pituitary–gonad axis, and ovarian function spermatocytes, spermatids and sperms in all (9, 10) Mated females treated by methoxsalen experimental groups compared to control require more time to become pregnant and group. mated males treated by methoxsalen need In addition, PUVA induces chromosomal more breeding attempts (12). This finding aberrations, sister chromatid exchanges, showed that the methoxsalen has effects on mutations, damage and cross-links in DNA of both males and females. Our results for human cells in vitro (20). In our study, experimental group I and II showed shrinkage methoxsalen alone and with PUVA showed and disorganize in seminiferous tubules and significant decrease in the mice body weight. release of primary spermatid cells, while there This decrease in the body weight showed that was not any change in group III. This result methoxsalen can act as an important indicated the effect of methoxsalen on repressor for cell division. Thus, the pituitary axis. Ruta graveolens L. (RG) interaction of PUVA with different parts of the contains various types of secondary cell such as DNA results in decreasing normal metabolites including coumarins, development. Investigation of the in vitro furanocoumarins (methoxsalen, xanthotoxin, interaction between DNA-methoxsalen in bergapten and isopimpinellin). The studies of darkness resulted in two mechanisms for RG showed a significant decrease in the DNA-methoxsalen binding: in low dosage number of spermatogonia type A and B, methoxsalen intercalates with DNA whereas primary spermatatocyte, spermatid, and in high dosage methoxsalen remains out of sperm in the experimental groups as a result the DNA molecule (21). Our result showed of methoxsalen (14). Although these results that intraperitoneal administration of 80mg/kg are compatible with our results, our data were of methoxsalen for two weeks results in obtained in lower dose and shorter time significant decrease in body weight and the (80mg/kg and 2 weeks) compared to RG number of spermatogenic cells specifically result (300 mg/kg and 100 days). It showed spermatids and spermatozoids. This finding that adverse effects of methoxsalen even start could be due to interaction between with low dose and in the short time. methoxsalen–DNA which leads to Another study showed that treated males chromosomal aberrations in low dosage and Wistar rats had significantly smaller pituitary short period of time. glands, fewer sperm per ejaculation, and Sufficient evidences for carcinogenicity of fewer sperm in the vasa defferentia and methoxsalen and UVA on experimental epididymis than control (9); methoxsalen has animals exist. Methoxsalen applied by oral, specific effect on sperm concentration and intraperitoneal administration and skin reproductive ability by inducing CREM gene application in combination with ultraviolet expression (13). Our results showed only radiation induced epidermal and dermal methoxsalen and PUVA decrease tumors in the mice (19). Increase in relative spermatogenic cells and body weight. Thus, testis weight and diameter of tunica albuginea effect of methoxsalen on pituitary–gonad axis only in experimental groups I and II could be could result in decrease of spermatogenic

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